Homeobox containing 1 (HMBOX1) is a novel transcription repressor that is significantly downregulated in human liver cancer tissues and cell lines, but the exact biological function of HMBOX1 in liver cancer is still unknown. We observed a negative association between HMBOX1 expression level and the clinical stages of liver cancer. HMBOX1 also increased the LC3 II/LC3 I ratio, the endogenous autophagy marker, and inhibited the p38/AKT/mTOR pathway. Furthermore, cancer stem cell specific genes, including CD133, KLF4, ESG1 and SOX2, were significantly downregulated upon HMBOX1 overexpression. Finally, the susceptibility of HepG2 cells to NK cell‑mediated cytolysis was increased by HMBOX1 overexpression and weakened by siRNA‑mediated inhibition of HMBOX1. All these findings indicated that HMBOX1 expression in hepatocytes could protect against the progression of liver cancer, and the underlying mechanisms may include promoting autophagy, inhibiting CSC phenotype and increasing the sensitivity of tumor cells to NK cell cytolysis. Therefore, HMBOX1 may be useful for developing new treatments for liver cancer.